Peptides suitable for use in antigen specific immunosuppressive therapy

ABSTRACT

This invention relates to peptides consisting of 16 to 55 amino acids, said peptides comprising at least one of the amino acid sequences LVCYYTSWS (SEQ ID NO:60), FLCTHIIYS (SEQ ID NO:61), IIYSFANIS (SEQ ID NO:62), LKTLLSVGG (SEQ ID NO:63), FIKSVPPFL (SEQ ID NO:64), FDGLDLAWL (SEQ ID NO:65), LYPGRRDKQ (SEQ ID NO:66), YDIAKISQH (SEQ ID NO:67), LDFISIMTY (SEQ ID NO:68), FISIMTYDF (SEQ ID NO:69), FRGQEDASP (SEQ ID NO:70), YAVGYMLRL (SEQ ID NO:71), MLRLGAPAS (SEQ ID NO:72), LAYYEICDF (SEQ ID NO:73), LRGATVHRT (SEQ ID NO:74), YLKDRQLAG (SEQ ID NO:75), LAGAMVWAL (SEQ ID NO:76), VWALDLDDF (SEQ ID NO:77) or LDLDDFQGS (SEQ ID NO:78). The peptides can be used in the treatment of T cell-mediated destruction of articular cartilage. Administration of pharmaceutical compositions based on these peptides can be used to induce systemic immunological tolerance to the autoantigens under attack of the autoreactive T-cells.

This application is a division of U.S. application Ser. No. 09/171,705,filed on Oct. 23, 1998, now U.S. Pat. No. 6,184,204 which is thenational stage of PCT/EP97/02051, filed on Apr. 22, 1997.

FIELD OF THE INVENTION

The invention relates to peptides and their use in treatment of chronicdestruction of articular cartilage in autoimmune diseases,pharmaceutical compositions comprising said peptides, a diagnosticmethod for the detection of autoreactive T cells in a test sample andtest kits to be used in said method.

BACKGROUND OF THE INVENTION

The immune system is established on a principle of discriminationbetween foreign antigens (non-self antigens) and autoantigens (selfantigens, derived from the individuals own body) achieved by a build intolerance against the autoantigens.

The immune system protects individuals against foreign antigens andresponds to exposure to a foreign antigen by activating specific cellssuch as T- and B lymphocytes and producing soluble factors likeinterleukins, antibodies and complement factors. The antigen to whichthe immune system responds is degraded by the antigen presenting cells(APCs) and a fragment of the antigen is expressed on the cell surfaceassociated with a major histocompatibility complex (MHC) class IIglycoprotein. The MHC-glycoprotein-antigen-fragment complex is presentedto a T cell which by virtue of its T cell receptor recognizes theantigen fragment conjointly with the MHC class II protein to which it isbound. The T cell becomes activated i.e. proliferates and/or producesinterleukines, resulting in the expansion of the activated lymphocytesdirected to the antigen under attack (Grey et al., Sci. Am., 261:38-46,1989).

Self antigens are also continuously processed and presented as antigenfragments by the MHC glycoproteins to T cells (Jardetsky et al., Nature353:326-329, 1991). Self recognition thus is intrinsic to the immunesystem. Under normal circumstances the immune system is tolerant to selfantigens and activation of the immune response by these self antigens isavoided.

When tolerance to self antigens is lost, the immune system becomesactivated against one or more self antigens, resulting in the activationof autoreactive T cells and the production of autoantibodies. Thisphenomenon is referred to as autoimmunity. As the immune response ingeneral is destructive, i.e. meant to destroy the invasive foreignantigen, autoimmune responses can cause destruction of the body's owntissue.

The contribution of T cells to autoimmune diseases has been establishedby several studies. In mice, experimental autoimmune encephalomyelitis(EAE) is mediated by a highly restricted group of T cells, linked bytheir specificity for a single epitope of myelin basic protein (MBP)complexed to an MHC class II molecule. In the Lewis rat, a species withhigh susceptibility to various autoimmune diseases, disease has beenshown to be mediated by T cells.

In humans autoimmune diseases are also thought to be associated with thedevelopment of auto-aggressive T cells. A destructive autoimmuneresponse has been implicated in various diseases such as rheumatoidarthritis (RA), in which the integrity of articular cartilage isdestroyed by a chronic inflammatory process. The mere presence ofcartilage appears necessary for sustaining the local inflammatoryresponse: it has been shown that cartilage degradation is associatedwith the activity of cartilage-responsive autoreactive T cells in RA(Sigall et al., Clin. Exp. Rheumat. 6:59, 1988; Glant et al., Biochem.Soc. Trans. 18:796, 1990; Burmester et al., Rheumatoid arthritis Smolen,Kalden, Maini (Eds) Springer-Verlag Berlin Heidelberg, 1992)Furthermore, removal of cartilage from RA patients by surgery was shownto reduce the inflammatory process. The cartilage proteins are thereforeconsidered to be target autoantigens which are competent of stimulatingT cells. Activation of these autoreactive T cells leads to developmentof autoimmune disease. Hence it can be anticipated that functionalelimination of these T cells could be beneficial in downregulation ofthe destructive autoimmune process. However, the identification of theautoantigenic components that play a role in the onset of rheumatoidarthritis has so far remained elusory.

The inflammatory response resulting in the destruction of the cartilagecan be treated by various drugs. However, these drugs areimmunosuppressive drugs that are nonspecific and have toxic sideeffects. The disadvantages of nonspecific immunosuppression makes this ahighly unfavorable therapy

Antigen-specific, nontoxic immunosuppression, such as for instancedescribed in WO-A-9510301, provides a very attractive alternative fornonspecific immunosuppression. The antigen-specific therapy involves thetreatment of patients with synthetic T cell-reactive peptides whichresemble or mimic the epitopes present on the autoantigen. Thesepeptides can therefore be used to induce systemic immunologicaltolerance, i.e. specific T cell tolerance, both to themselves and to theautoantigen. The induced systemic immunological tolerance is based onthe long-observed phenomenon that animals which have been fed or haveinhaled an antigen or epitope are less capable of developing a systemicimmune response towards said antigen or epitope when said antigen orepitope is introduced via a systemic route. To effectively use thepeptide-induced systemic tolerance therapy to treat the T cell mediatedcartilage destruction, there is no great need for T cell-reactivepeptides which can desensitize patients against the self antigen that isactivating the T cells responsible for the inflammatory process.

It is an object of the invention to provide peptides which are able toinduce systemic immunological tolerance, more in particular specific Tcell tolerance, to the responsible cartilage antigen in patientssuffering from T cell-mediated cartilage destruction. It is anotherobject of the invention to provide a method for detecting autoreactive Tcells involved in the destruction of articular cartilage and test kitsto be used in said method.

The present invention provides for such peptide.

In a first aspect of the invention there is provided for peptidesconsisting of 16 to 55 amino acid residues, said peptide comprising atleast one of the amino acid sequences LVCYYTSWS (SEQ ID NO:60),FLCTHIIYS (SEQ ID NO:61), IIYSFANTS (SEQ ID No:62), LKTLLSVGG (SEQ IDNO:63), FIKSVPPFL (SEQ ID NO:64), FDGLDLAWL (SEQ ID NO: 65), LYPGRRDKQ(SEQ ID No:66), YDIAKISQH (SEQ ID NO:67), LDFISIMTY (SEQ ID NO:68),FISIMTYDF (SEQ ID NO:69), FRGOEDASP (SEQ ID NO:70), YAVGYMLMRL, (SEQ IDNO:71), MLRLGAPAS (SEQ ID NO:72). LAYYEICDF (SEQ ID NO:73), LRGATVHRT(SEQ ID NO:74), YTKDRQLAG (SEQ ID NO:75). LAGAVWAL (SEQ ID NO:76),VWALDLDDF (SEQ ID NO:77) or LDLDDFQGS (SEQ ID NO:78).

In particular, the peptide according to the invention comprises at leastone of the amino acid sequence YKLVCYYTSWSQYREG (SEQ ID NO:1),YTSWSQYREGDGSCFP (SEQ ID NO:2), LDRFLCTHIIYSFANI (SEQ ID NO:5),THIIYSFANISNDHID (SEQ ID NO:6), NLTLLSVGWNFGS (SEQ ID NO:12),NTQSRRTFIKSVPPFL (SEQ ID NO:16), TFIKSVPPFLRTHGFD (SEQ ID NO:17),PPFLRTHGFDGLDLAW (SEQ ID NO:18), HGFDGLDLAWLYPGRR (SEQ ID NO:19),DLAWLYPGRRDKQHFT (SEQ ID NO:20), TIDSSYDIAKISQHLD (SEQ ID NO:28),DIAKISQHLDFISIMT (SEQ ID NO:29), QHLDFISIMTYDPHGA (SEQ ID NO:30),SPLFRGQEDASPDRFS (SEQ ID NO:34), DYAVGYMLRLGAPASK (SEQ ID NO:37),MLRLGAPASKLVMGIP (SEQ ID NO:38), PASKLVMGIPTFGRSF (SEQ ID NO:39),GTLAYYEICDFLRGAT (SEQ ID NO:46), EICDFLRGATVHRTLG (SEQ ID NO:47),RGATVHRTLGQQVPYA (SEQ ID NO:48), VKSKVQYLKDRQLAGA (SEQ ID NO:53),YLKDRQLAGAMVWALD (SEQ ID NO:54), LAGAMVWALDLDDFQG (SEQ ID NO:55),WALDLDDFQGSFCGQD (SEQ ID NO:56) or DFQGSFCGQDLRFPLT (SEQ ID NO:57).

Preferably, the peptide according to the present invention comprises oneof the amino acid sequences YKLVCYYTSWSQYREG (SEQ ID NO:1),YTSWSQYREGDGSCFP (SEQ ID NO: 2) LDRFLCTHIIYSFANI (SEQ ID NO:5),THIIYSFANISNDHID (SEQ ID NO 6), PNLKTLLSVGGWNFGP (SEQ ID NO:12),QHLDFISIMTYDFHGA (SEQ ID NO:30), SPLFRGOEDASPDRPS (SEQ ID No:34),DYAVGYMLRLGPASK (SEQ ID NO:37), MLRLGAPASKLVMGIP (SEQ ID NO:38),YLKDRQLAGAMVWALD (SEQ ID NO:54) or LAGAMVWALDLDDFQG (SEQ ID NO:55).

More preferably, the peptide according to the invention comprises one ormore of the amino acid sequences YTSWSQYREGDGSCFP (SEQ ID NO:2),SPLFRGQEDASPDRFS (SEQ ID NO:34), MLRLGAPASKLVMGIP (SEQ ID NO:38),YLKDRQLAGAMVWALD (SEQ-ID NO:54) or LAGAMVWALDLDDFQG (SEQ ID NO:55).

The peptides according to the invention consist of 16 to 55, preferably16 to 35, more preferably 16, to 25, most preferably 16 amino acidresidues.

Highly preferred peptides according to the invention arehexadecapeptides consisting of the amino acid sequence YKLVCYYTSWSQYREG(SEQ ID NO:1) YTSWSQYREGDGSCFP (SEQ ID NO:2) LDRFLCTHIIYSFANI (SEQ IDNO:5), THIIYSFANISNDHID (SEQ ID NO 6). PNLKTLLSVGGWNFGS (SEQ ID NO:12),QHLDFISIMTYDFHGA (SEQ ID NO:30), SPLFRGQEDASPDRFS (SEQ ID NO:34),DYAVGYMLRLGAPASK (SEQ ID NO:37), MLRLGAPASKLVMGIP (SEQ ID NO:38),YLKDRQLAGAMVWALD (SEQ ID NO:54) or LAGAMVWALDLDDFQG (SEQ ID NO:55), morein particular the amino acid sequences YTSWSQYREGDGSCFP (SEQ ID NO:2),SPLFRGQEDASPDRFS (SEQ ID NO:34), MLRLGAPASKLVMGIP (SEQ ID NO:38),YLKDRQLAGAMVWALD (SEQ ID NO:54) or LAGAMVWALDLDDFQG (SEQ ID NO:55)

Also within the scope of the invention are multimers of the peptidesaccording to the invention such as for example a dimer or trimer of thepeptides according to the invention A multimer according to theinvention can either be a homomer, consisting of a multitude of the samepeptide, or a heteromer consisting of different peptides.

The characteristic amino acid sequences of the peptides according to theinvention can be flanked by random amino acid sequences. Preferred areflanking sequences, that have a stabilizing effect on the peptides, thusincreasing their biological availability.

The present invention is based on the unexpected discovery, that HumanCartilage glycoprotein 39 (herein after referred to as HC gp-39) is atarget autoantigen in RA patients which activates specific T cells, thuscausing or mediating the inflammatory process. HC gp-39 derived peptideswere predominantly recognized by autoreactive T cells from RA patientsbut rarely by T cells from healthy donors, thus indicating that HC gp-39is an autoantigen in RA. The arthritogenic nature of HC gp-39 wasfurther substantiated in the Balb/c mouse. A single, subcutaneousinjection of said protein in Balb/c mice was able to initiate arthriticsigns in the animals. The course of the HC gp-39-induced disease wascharacterized by relapses occurring periodically in fore paws and/orhind paws and gradually developed from a mild arthritis into a moresevere form. Also, a symmetrical distribution of afflicted joints wasobserved which is, together with the observation of recurrent relapsesand nodule formation, reminiscent of disease progression in arthritis,especially RA.

Even more surprisingly it was found that administration of HC gp-39resulted in immunological tolerance and, more importantly, in delayedand/or suppressed arthritic development.

The amino acid sequences given in SEQ ID NO's 60-78, more specificallythe sequences given in SEQ ID NO's 1, 2, 5, 6, 12, 16-20, 28-30, 34,37-39, 46-48, 53 -57 resemble MHC class II restricted T cell epitopeswhich are present on HC gp-39. Thus, the peptides according co theinvention can also be understood to encompass fragments of theautoantigen HC gp-39 which comprise one or more of the above identifiedMHC Class II restricted T-cell epitopes and they are also within thescope of the invention.

Although HC gp-39 was disclosed in Hakala et al., J. Biol. Chem., Vol.268, No. 34, 25803 (1993), in which it was described as a chitinaseprotein and suggested for use as a suitable marker for rheumatoidarthritis, any hint or suggestion towards the arthritogenic nature of HCgp-39 was absent.

The peptides according to the invention can be prepared by well knownorganic chemical methods for peptide synthesis such as, for example,solid-phase peptide synthesis described for instance in J. Amer. Chem.Soc. 85.2149 (1963) and Int. J. Peptide Protein Res. 35:161-214 (1990).

The peptides according to the invention can also red by recombinant DNAtechniques. A nucleic acid sequence coding for a peptide according tothe invention or a multimer of said peptides is inserted into anexpression vector. Suitable expression vectors are, amongst others,plasmids, cosmids, viruses and YAC's (Yeast Artificial Chromosomes)which comprise the necessary control regions for replication andexpression. The expression vector can be brought to expression in a hostcell. Suitable host cells are, for instance, bacteria, yeast cells andmammalian cells. Such techniques are well known in the art, see forinstance Sambrooke et al, Molecular Cloning: a Laboratory Manual, ColdSpring Harbor laboratory Press, Cold spring Harbor, 1989.

The peptides according to the invention are T-call reactive peptides,which are recognized by and are able to stimulate activated,autoreactive T-cells. These autoreactive T cells are found in the bloodof RA patients but rarely ill healthy donors.

Thus, according to the invention the synthetic peptides, said peptidesresembling the MHC Class II restricted W-cell epitopes present on thetarget autoantigen HC gp-39, are very suitable for use in a therapy toinduce specific T-cell tolerance to HC gp-39 in mammals, morespecifically humans, suffering from T-cell mediated cartilagedestruction, such as for example arthritis, more specifically rheumatoidarthritis.

Although WO 95/01995 and WO 95/02188 describe the diagnostic use of HCgp-39 as a marker for RA, the arthritogenic nature of HC gp-39 isneither disclosed nor suggested. Nowhere do they hint or suggest towardsthe use of fragments of HC gp-39 or T-cell reactive peptides accordingto the present invention in the antigen or peptide specific therapy toinduce T-cell specific tolerance to the HC gp-39 in the cartilage underattack.

According to the invention, patients suffering from T-cell mediateddestruction of the articular cartilage can be treated with atherapeutical composition comprising one or more peptides according tothe invention and a pharmaceutical acceptable carrier. Administration ofthe pharmaceutical composition according to the invention will inducesystemic immunological tolerance, in particular tolerance of thespecific autoreactive T cells of these patients, to the autoantigenicproteins in the articular cartilage under attack and other self antigenswhich display the identified MHC Class II binding T cell epitopescharacterized or mimiced by the amino acid sequences of one or more ofthe peptides according to the invention. The induced tolerance thus willlead to a reduction of the local inflammatory response in the articularcartilage under attack.

Very suitable peptides to be used in a pharmaceutical compositionaccording to the invention are the peptides having 16-55, preferably16-35, more preferably 16-25, most preferably 16 amino acid residues,said peptides comprising at least one of the amino acid sequencesLVCYYTSWS (SEQ ID NO:60)FLCTHIIYS (SEQ ID NO:61), IIYSFANIS (SEQ IDNO:62), LKTLLSVGC (SEQ ID NO:63), FIKSVPPFL (SEQ ID NO:64), FDGLDLAWL(SEQ ID NO: 65), LYPGRRDKQ (SEQ ID NO:66), YDIAKISQH (SEQ ID NO:67),LDFISIMTY (SEQ ID NO:68), FISIMTYDF (SEQ ID NO:69). FRGOEDASP (SEQ IDNO:70), YAVGYMLRL (SEQ ID NO:71), MLRLGAPAS (SEQ ID NO:72), LAYYEICDF(SEQ ID NO:73), LRGATVHRT (SEQ ID NO:74), YLKDRQLAG (SEQ ID NO:75),LAGAMVWAL (SEQ ID NO:76), VWALDLDDF (SEQ ID NO:77) or LDLDDFQGS (SEQ IDNO:78) more in particular one of the amino acid sequencesYCLVCYYTSWSQYREG (SEQ ID NO:1), YTSWSSOYREGDGSCFP (SEQ ID NO: 2),LDRFLCTHIIYSFANI (SEQ ID NO:5), THIISYSFANISNDHID (SEQ ID NO:6),PNLKTLLSVGGWNFGS (SEQ ID NO:12) NTQSRRTFIKSVPPFL (SEQ ID NO:16),TFIKVPPFLRTHFGD (SEQ ID NO:17), PPFLRTHGFDGLDLAW (SEQ ID NO:18),HGFDGLDLAWYPGRR (SEQ ID NO:19), DLAWLYPGRRDKQHFT (SEQ ID NO:20),TIDSSYDIAKISQHLD (SEQ ID NO:28), DIAKISQHLDFISIMT (SEQ ID NO:29),QHLDFISIMTYDFHCGA (SEQ ID NO: 30), SPLFRGOEDASPORRFS (SEQ ID NO:34),DYAVGYMLRLGAPASK (SEQ ID NO:37), MLRLGAPASKLVMGIP (SEQ ID NO:38),PASKLVMGIPTFGRSF (SEQ ID NO:39), GTLAYYEICDFLRGAT (SEQ ID NO:46),EICDFLRGATVHRTLG (SEQ ID NO:47), RGATVHRTLGQQVPYA (SEQ ID NO:48),VKSKVQYLKDRQLAGA (SEQ ID NO:53), YLKDRQLAGAMVWALD (SEQ ID NO:54),LAGAMVWALDLDDFQG (SEQ ID NO:55), WALDLDDFQGSFCGQD (SEQ ID NO:56) orDFQGSFCGQDLRFPLT (SEQ ID NO:57).

Specifically preferred in a pharmaceutical composition according to theinvention are the peptides having 16-55, preferably 16-35, morepreferably 16-25, most preferably 16 amino acid residues, said peptidescomprising at least one of the amino acid sequences YKLVCYYTSWSQYREG(SEQ ID NO:1), YTSWSQYREGDGSCFP (SEQ ID NO:2), LDRFLCTHIIYSFANI (SEQ IDNO:5), THIIYSFANISNDHID (SEQ ID NO:6), PNLKTLLSVGGWNFGS (SEQ ID NO:12),QHLDFISIMTYDFHGA (SEQ ID NO:30), SPLFRGQEDASPDRFS (SEQ ID NO:34),DYAVGYMLRLIGAPASK (SEQ ID NO:37), MLRLGAPASKLVMGIP (SEQ ID NO:38),YLKDRQLAGAMVWALD (SEQ ID NO:54) or LAGAMVWALDLDDFQG (SEQ ID NO:55).

Highly preferred in a pharmaceutical composition according to theinvention are peptides having 16-55, preferably 16-35, more preferably16-25, most preferably 16 amino acid residues, said peptides comprisingat least one of the amino acid sequences YTSWSQYREGDGSCFP (SEQ ID NO:2),SPLFRGQEDASPDRFS (SEQ ID NO:34), MLRLGAPASKLVMGIP (SEQ ID NO:38),YLKDRQLAGAMVWALD (SEQ ID NO:54) or LAGAMVWALDLDDFQG (SEQ ID NO:55).

Most preferred in a pharmaceutical composition according to theinvention are hexadecapeptides consisting of the amino acid sequenceYKLVCYYTSWSQYREG (SEQ ID NO:1) YTSWSQYREGDSGFP (SEQ ID. NO:2),LDRFLCTHIIYSFANI (SEQ ID NO:5), THIIYSFANISNHID (SEQ. ID NO:6),PNLKTLLSVGGWNFGS (SEQ ID NO:12), QHLDFISIMTYDFHGA (SEQ ID NO:30),SPLFRGQEDASPDRFS (SEQ ID NO:34), DYAVGYMLRLGAPASK (SEQ ID NO:37),MLRLGAPASKLVMGIP (SEQ ID NO:38), YLKDRQLAGAMVWALD (SEQ ID NO:54) orLAGAMVWALDLDDFQG (SEQ ID NO:55), more in particular the amino acidsequences YTSWSQYREGDGSCFP (SEQ ID NO:2). SPLFRGQEDASPDRFS (SEQ IDNO:34), MLRLGAPASKLVMGIP (SEQ ID NO:38), YLKDRQLACAMVWALD (SEQ ID NO:54)or LAGAMVWALDLDDFQG (SEQ ID NO:55).

The peptides according to the invention have the advantage that theyhave a specific effect on the autoreactive T cells thus leaving theother components of the immune system intact as compared to thenonspecific suppressive effect of immunosuppressive drugs. Treatmentwith the peptides according to the invention will be safe and no toxicside effects will occur.

Systemic immunological tolerance can be attained by administering highor low doses of peptides according to the invention. The amount ofpeptide will depend on the route of administration, the time ofadministration, the age of the patient as well as general healthconditions and diet.

In general, a dosage of 0.01 to 1000 μg of peptide per kg body weight,preferably 0.5 to 500 μg, more preferably 0.1 to 100 μg of peptide canbe used.

Pharmaceutical acceptable carriers are well known to those skilled inthe art and include, for example, sterile salin, lactose, sucrose,calcium phosphate, gelatin, dextrin, agar, pectin, peanut oil, olive oilsesame oil and water. Other carriers may be, for example MHC class IImolecules, if desired embedded in liposomes.

In addition the pharmaceutical composition according to the inventionmay comprise one or more adjutants. Suitable adjutants include, amongstothers, aluminum hydroxide, aluminum phosphate, amphigen, tocophenols,monophosphenyl lipid A, muramyl dipeptide and saponins such as Quill A.Preferably, the adjutants to be used in the tolerance therapy accordingto the invention are mucosal adjutants such as the cholera toxineB-subunit or carbomers, which bind to the mucosal epithelium. The amountof adjuvant depends on the nature of the adjuvant itself.

Furthermore the pharmaceutical composition according to the inventionmay comprise one or more stabilizers such as, for example, carbohydratesincluding sorbitol, mannitol, starch, sucrosedextrin and glucose,proteins such as albumin or casein, and buffers like alkalinephosphates.

Suitable administration routes are intramuscular injections,subcutaneous injections, intravenous injections or intraperitonealinjections, oral administration and nasal sprays.

The peptides according to the invention are also very suitable for usein a diagnostic method to detect the presence of activated autoreactiveT cells involved in the chronic inflammation and destruction of thearticular cartilage.

The diagnostic method according to the invention comprises the followingsteps:

-   -   a) isolation of the peripheral blood mononuclear cells (PBMC)        from a blood sample of an individual,    -   b) culture said PBMC under suitable conditions,    -   c) incubation of said PBMC culture in the presence of one or        more peptides according to the invention, and    -   d) detection of a response of T cells, for example a        proliferative response, indicating the presence of activated        autoreactive T cells in the individual.

The detection of a proliferative response of T cells can be detected by,for example, the incorporation of ³H-thymidine.

Also within the scope of the invention are test kits which comprise oneor more peptides according to the, invention. These test kits aresuitable for use in a diagnostic method according to the invention.

The following examples are illustrative for the invention and should inno way be interpreted as limiting the scope of the invention.

EXAMPLES

Methods

Patients

This study included 7 DR4(DRB1*0401)-positive patients diagnosed assuffering from RA according to the ARA criteria (Arnett et al., (1988),Arthritis Rheum. 31, 315). Peripheral blood samples were obtained withinformed consent. There were five women and two men aged 46-79 years.Their duration of disease ranged from over 10 to over 30 years. Threeout of 7 patients had at least 3 swollen joints. Four patients did notshow any signs of active disease. All patients were on medication: Fourpatients were treated with prednisone, three patients receivedanti-rheumatic agents and 4 patients were treated with NSAID's as well.

Peripheral blood samples from 5 healthy donors carrying theDR4(DRB1*0401) specificity were obtained with informed consent andincluded in this study as a control.

Definition of HLA-DR Polymorphisms.

Patient and healthy donor peripheral blood mononuclear cells (PBMC)isolated from heparinized peripheral blood by standard centrifugation onFicoll-Paque were stimulated with PHA (Welcome, Dartford, UK) to obtain5×10⁶−10⁷ lymphocytes. The QIA amp blood kit (QIAGEN Inc,) was used topurify chromosomal DNA from cultured cells according to themanufacturers instruction. Chromosomal DNA extracts were analysed usinga DR ‘low resolution’ SSP kit. DR4 subtyping was performed using theDynal DRB1*04-SSP kit. MHC DR typing was performed at the TransparentSerology Laboratory, University Hospital, Nijmegen, The Netherlands.

TABLE I RA Patient stage synovitis duration HLA-DR 191 IV no >30 years0401/01 259 III-IV yes >30 years 0401/16 262 III-IV yes >10 years 0401/0408 272 III-IV no >30 years  0401/0701 276 IV no >30 years0401/14 286 IV no   20 years  0401/0408 287 III-IV yes   20 years0401/13 HD HLA-DR 155 0401/14 157 0401/13 168 0401/07 230 0401/07 2350401/13

Peptide Synthesis

Peptides were synthesized at Eurosequence (Groningen. The Netherlands).Peptides were synthesized from the C-terminus to the N-terminus on a 10μmol scale using solid-phase FMOC chemistry. The crude peptides werepartly purified by several other preparations. As indicated by themanufacturer, at least 35% of the lyophilized product contained thedesired full length product. The rest contained salt and remainingsolvent. The quality of the final product was checked by sequenceanalysis, amino acid analysis and/or RF-HPLC. The sequences of thepeptides synthesized are enlisted in Table II.

TABLE II Amino acid sequences of the peptides used in this study. SEQ IDNO: residu peptide 1 22-37 YKLVCYYTSWSQYREG 2 28-43 YTSWSQYREGDGSCFP 334-49 YREGDGSCFPDALDRF 4 40-55 SCFPDALDRFLCTHII 5 46-61 LDRFLCTHIIYSFANI6 52-67 THIIYSFANISNDHID 7 58-73 FANISNDHIDTWEWND 8 64-79DHIDTWEWNDVTLYGM 9 70-85 EWNDVTLYGMLNTLKN 10 76-91 LYGMLNTLKNRNPNLK 1182-97 TLKNRNPNLKTLLSVG 12  88-103 PNLKTLLSVGGWNFGS 13  94-109LSVGGWNFGSQRFSKI 14 100-115 NFGSQRFSKIASNTQS 15 106-121 FSKIASNTQSRRTPIK16 112-127 NTQSRRTFIKSVPPFL 17 118-133 TFIKSVPPFLRTHGFD 18 124-139PPFLRTHGFDGLDLAW 19 130-145 HGFDGLDLAWLYPGRR 20 136-151 DLAWLYPGRRDKQHFT21 142-157 PGRRDKQHFTTLIKEM 22 148-163 QHFTTLIKEMKAEFIK 23 154-169IKEMKAEFIKEAQPGK 24 160-175 EFIKEAQPGKKQLLLS 25 166-181 QPGKKQLLLSAALSAG26 172-187 LLLSAALSAGKVTIDS 27 178-193 LSAGKVTIDSSYDIAK 28 184-199TIDSSYDIAKISQHLD 29 190-205 DIAKISQHLDFISIMT 30 196-211 QHLDFISIMTYDFHGA31 202-217 SIMTYDFHGAWRGTTG 32 208-223 FHGAWRGTTGHHSPLF 33 214-229GTTGHHSPLFRGQEDA 34 220-235 SPLFRGQEDASPDRFS 35 226-241 QEDASPDRFSNTDYAV36 232-247 DRFSNTDYAVGYMLRL 37 238-253 DYAVGYMLRLGAPASK 38 244-259MLRLGAPASKLVMGIP 39 250-265 PASKLVMGIPTFGRSF 40 256-271 MGIPTFGRSFTLASSE41 262-277 GRSFTLASSETGVGAP 42 268-263 ASSETGVGAPISGPGI 43 274-289VGAPISGPGIPGRFTK 44 280-295 GPGIPGRFTKEAGTLA 45 286-301 RFTKEAGTLAYYEICD46 292-307 GTLAYYEICDFLRGAT 47 298-313 EICDFLRGATVHRTLG 48 304-319RGATVHRTLGQQVPYA 49 310-325 RTLGQQVPYATKGNQW 50 316-331 VPYATKGNQWVGYDDQ51 322-337 GNQWVGYDDQESVKSK 52 328-343 YDDQESVKSKVQYLKD 53 334-349VKSKVQYLKDRQLAGA 54 340-355 YLKDRQLAGAVVWALD 55 346-361 LAGAMVWALDLDDFQG56 352-377 WALDLDDFQGSFCGQD 57 358-373 DFQGSFCGQDLRFPLT 58 364-379CGQDLRFPLTNAIKDA 59 368-383 LRFPLTNAIKDALAATPeptide HLA-DR Binding Assay

DR4 (DRB1*0401) and DR4 (DRB1*0404) molecules were purified from thehomozygous EBV-transformed human B lymphoblastoid cell lines Huly138IC2and BM92 using the mAb t243, directed against a monomorphic determinanton the DR-complex (Lampson, L. A and R. Levy (1980), J. Immunol. 125:293299).

The peptide binding studies were performed using a semi-quantitativecompetition binding assay (Joosten et al 1994, Int. Immunol. 6, 751).Briefly, purified HLA-DR molecules (30 nM DR4(DRB1*0401) or 15 nM DR4(DRB1*0404) were incubated at pH5.0 with 50 nM biotinylated markerpeptide (HA 309_(y→F)) and a concentration range of competitor peptidein a final volume of 25 μl binding buffer (PBS containing 0.01% Na₃,0.05% NP-40, 5% DMSO, 1 mM AEBSF, 1 mM N-ethyl maleimide, 8 mM EDTA and10 μm pepstatin A). After 44 hr of incubation at RT, HLA DR bound markerpeptide was separated from free marker peptide using a 96 well vacuumdotblot apparatus (Hybri. dot, BRL) and a nitrocellulose membrane(Hybond ECL, Amersham, UK). The nitrocellulose filters were blocked with0.5% DNA blocking reagent (Boehringer) in 0.1 M maleic acid, 150 mMNaCl, pH7.5.

After 0.5-1 hr, the filters were washed in PBS, 0.05% Tween 20 (Sigma)and incubated with Strepcavidin-HRPO (Southern Biotechnology) in a1:10.000 dilution. Biotinylated peptides were detected by enhancedchemiluminescence using a Western Blot ECL kit (Amersham). Exposure ofthe preflashed films (Hyperfilm-ECL, Amersham) was for 10 min. The spotswere analysed by scanning the films and using Image Quant/Excel softwarefor analysis.

The affinity of a given peptide for binding DRB1*0401-encoded moleculeswas related to competition with the marker peptide. This relativebinding affinity was defined as the peptide concentration at which thesignal was reduced to 50% (IC₅₀).

Proliferative Responses of Blood Mononuclear Cells

In order to identify T-cell epitopes within HC gp-39, 59 peptides of 16AA in length, overlapping by 10 AA were tested for their capacity toinduce a proliferative response in PBMC from RA patients and healthycontrols carrying the DR4 (DRB1*0401) specificity (Table 1). Table 2enlists the sequences of the peptides tested.

PBMC obtained from heparinized venous peripheral blood were isolated bystandard centrifugation on a Ficoll-Paque gradient. Cells were culturedin four-fold at a concentration of 1,5×10⁵ cells/well in mediumsupplemented with 10% heat-inactivated, autologous plasma, L-glutamine,2-ME and antibiotics in flatbottomed microtiter plates. Cells wereincubated in medium alone or in the presence of phytohaemagglutinin(PHA) (2:5 μg/ml) to assert cell viability, or in the presence of 10 or100 μg/ml of the HC gp-39-derived peptides. In several cases, sets of 2or 3 sequential peptides were tested due to limited PBMC numbers ofindividual donors. Cultures were incubated in a total volume of 210 μlfor 7 days at 370° C. in a humidified atmosphere of 5% CO₂. Cultureswere pulsed during the last 18 hours with 0.25 μCi ³H-thymidine([³H]TdR). Cells were harvested on glassfiber filters and [³H] TdRincorporation was measured by gas scintillation (Packard Matrix 96βcounter). Only peptides inducing a proliferative response at both 10and 100 μg/ml were considered to contain a T-cell epitope. Responseswere defined positive if stimulation index values (SI, antigen-specificcounts per 5 min (cp5 m)/background cp5 m) exceeded or equaled 2.

Results

Identification of T-Cell Epitopes by Proliferative Responses of BloodMononuclear Cells

T-cell reactivity to HC gp-39-derived peptide as analyzed by measuringthe PBMC proliferative response in DR4 (DRB1*0401)-positive RA patientsand healthy donors. Proliferative responses were tested in autologousplasma. In Table IIIA and IIIB the results of 7 experiments arepresented showing the responses of RA patients (Table IIIA) and theresponses of healthy donors (Table IIIB) to 59 overlapping sequencesderived from HC gp-39. Donors found to respond to both concentrations(100 and 10 μg/ml) of a peptide were ranked as responders and donorswhich did not respond to both concentrations tested were ranked asnon-responders (NR).

Responses to the individual peptides 1, 2, 5, 6, 12, 15, 30, 34, 37, 38,40, 41, 54 and 55 (the numbers respond to the respective SEQ ID NO ofeach peptide, for example, peptide 30 means: peptide having amino acidsequence of SEQ ID NO:30) were observed in one or more donors, therebyidentifying these sequences as T-cell epitopes.

Interestingly, responses to peptides 2, 34, 38, 40, 54 and 55 wereobserved in RA patients only.

On the other hand, peptides 12 and 41 induced only responses in healthydonors (230, 235) thus far.

In addition, as can be seen in Table 3, responses were found to thefollowing sets of: peptides 1/2, 1/2/3, 4/5/6, 5/6, 15/16, 17/18, 19/20,28/29/30, 29/30, 37/38, 37/38/39, 39/40, 46/47/48, 53/54, 55/56 and55/56/57. These results are in accordance with most of the results ofthe individual peptides mentioned above. Moreover, the responses againstthe sets of peptides define regions that contain additional T-cellepitopes, i.e. the regions covered by peptides 16-20 (residu 112-151),28-29 (residu 184-205), 38-40 (residu 244-271), 46-48 (residu. 292-319)and 53-57 (334-373).

Six out of 7 DR4 (DRB1*0401)-positive RA patients responded to HCgp-39-derived peptides or sets of peptides and were therefore ranked asresponders. In the healthy donor group (HD), 3 out 5 donors were rankedas responders. In general, RA patients appeared to respond to many moreHC gp-39 regions than healthy donors (healthy donor 230 being anexception) For example, PBMC from RA patient 272, which were testedagainst individual peptides, appeared to respond to a total of 11peptides (1, 2, 5, 6, 30, 34, 37, 38, 40, 54 an 55). PBMC of the otherpatients (patient 287 being an exception) showed responses against setsof peptides overlapping these 11 sequences and identified someadditional regions containing T-cell epitopes (peptides 14-20 and46-48).

PBMC derived from a healthy donor (230) further confirmed the presenceof T-cell epitopes in peptides 1, 5, 6, 15, 30 and 37.

Overall, the peptides or sets of peptides most frequently recognizedcontain peptides 1/2, 5/6, 30, 37/38, 54/55).

Correlation of T-cell Epitopes and DRB4 (DRB1*0401)Binding

Peptides 1, 2, 5, 6, 12, 15, 30, 34, 37, 38, 40, 41, 54 and 55 were allfound to stimulate peripheral blood derived T-cells. As a corrollary tothis finding, all of these peptides were found to bind to DR4(DRB1*0401) with relatively high affinity (except peptides 2 and 38which bind with intermediate relative affinity). Peptides 3, 4, 16, 17,18, 19, 20, 28, 29, 39, 46, 47, 48, 53, 56 and 57 were tested in setsrather than individually. It is very likely that several of thesepeptides also contain relevant T-cell epitopes. In any case, thesepeptides all can-bind DRB4 (DRB1*0401) with high to intermediaterelative affinity (except for peptide 20 which binds with poor relativeaffinity).

TABLE IIIA Peptide-induced proliferative responses of PBMC from RApatients. RA: 272 262 276 286 191 287 259 0401 0401 0401 0401 0401 04010401 0401 peptide R R R R R NR R binding 1 pos +++ 2 pos pos pos pospos + 3 + 4 + 5 pos pos +++ 6 pos pos pos +++ 7 8 9 10 11 12 13 14& 15&+++ 16 pos +++ 17 ++ 18 pos + 19 + 20 pos +/− 21 22 23 24 25 26 27 28+++ 29 pos +++ 30 pos pos pos pos +++ 31 32 33 34 pos +++ 35 36 37 pos+++ 38 pos pos pos pos pos + 39 ++ 40& pos pos +++ 41& 42 43 44 45 46+++ 47 pos +++ 48 + 49 50 51& 52& 53 +++ 54 pos pos pos +++ 55 pos +++56 pos pos +++ 57 ++ 58 59 BG 0.2 0.7 0.5 0.8 2.4 0.9 0.2pos=positive responses to both 100 and 10 microgram/ml of peptide orsets of peptides (SI≧2 were regarded positive). Together the pep tides(16 AA in length and overlapping by 10 AA) cover the complete maturesequence of mature HC gp-39 (residu 22-383). Peptides were synthesizedat Eurosequence (Groningen, The Netherlands). RA=rheumatoid arthritispatient. 0401=donor carrying the RA associated HLA-DR1*0401 specificity.NR=non-responder. R=responder. BG=mean of background counts per 5minutes×10 measured in wells without antigen. +++=high affinity binder(IC50<1 μM), ++=good affinity binder (1<IC50<10 μM); +=intermediatebinder (10<IC50<100 μM); +/−=poor binder (100<IC50<1000 μM);−=non-binder (IC50>1000 μM)

TABLE IIIB Peptide-induced proliferative responses of PBMC from healthydonors. HD 155 157 168 230 235 0401 HD 0401 0401 0401 0401 0401 0401peptide R NR NR R R binding  1 pos +++  2  3  4  5 pos +++  6 pos +++  7 8  9 10 11 12 pos +++ 13   14&   15& pos +++ 16 17 18 19 20 21 22 23 2425 26 27 28 29 30 pos +++ 31 32 33 34 35 36 37 pos pos pos +++ 38 39 4041 pos +++ 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 BG 4, 210, 4 2, 2 3, 6 3, 5pos=positive responses to both 100 and 10 microgram/ml of peptide orsets of peptides (SI>2 were regarded positive). Together the peptides(16 AA in length and overlapping by 10 AA) cover the complete maturesequence of mature HC gp-39 (residu 22-383). Peptides were synthesizedat Eurosequence (Groningen, The Netherlands). HD=healthy donor.0401=donor carrying the RA-associated HLA-DRB1*0401 specificity.NR=non-responder. R=responder. BG=mean of background counts per 5minutes×10⁻³ measured in wells without antigen. +++=high affinity binder(IC50<1 μM); ++=good affinity binder (1<IC50<10 μM); +=intermediatebinder (10<IC50<100 μM); +/−=poor binder (100<IC50<1000 μM);−=non-binder (IC50>1000 μM)Abbreviations

-   AEBSF: 4-(2-AminoEthyl)-BenzeneSulfonyl Fluoride-   BB: binding buffer-   BCA: Bicinchoninic Acid-   BSA: bovine serum albumin-   DMSO: Dimethyl Sulfoxide-   ECL: Enhanced Chemiluminescence-   EDTA: EthyleneDiamine Tetra Acetic acid-   FACS: Fluorescence Activated Cell Sorter-   HLA: Human Leukocyte Antigens-   HPLC: High Pressure Liquid Chormtatography-   HRP: Horse Radish Peroxidase-   MHC CLASS II: Major Histocompatibility Complex class II-   NMR: Nuclear Magnetic Resonance-   NP-40: Nonidet P-40-   PBS: Phosphate Buffered Saline-   PVDF: Polyvinylidene difluoride-   RA: Rheumatoid Arthritis-   SDS-PAGE: Sodium DodecylSulfate Polyacrylamide Gel Electrophoresis

1. A peptide consisting of 16 to 55 amino acid residues, comprising theamino acid sequence YKl VCYYTSWSQYREG (SEQ ID NO: 1).
 2. Apharmaceutical composition comprising a peptide according to claim 1,and a pharmaceutically acceptable carrier.
 3. A test kit for use in thedetection of activated autoreactive T cells, comprising a peptideaccording to claim
 1. 4. A pharmaceutical composition consisting of oneor more peptides selected from the group consisting of peptidesconsisting of 16 to 55 amino acid residues and comprising the amino acidsequence YKLVCYYTSWSQYREG (SEQ ID NO:1).
 5. A method of inducingsystemic immunological tolerance, comprising administering mucosally toa patient in need thereof a pharmaceutical composition comprising one ormore peptides containing 16 to 55 amino acid residues selected from thegroup consisting of the amino acid sequence LVCYYTSWS (SEQ ID NO:60) anda pharmaceutically acceptable carrier.
 6. A method for inducing asystemic immunological tolerance, comprising administering muscosally toa patient in need thereof a pharmaceutical composition according toclaim 4.